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Fluconazole (2-(2,4-difluorophenyl)-1,3-bis(1 H -1,2,4-triazol-1-yl)propan-2-ol), which was patented in 1981 and introduced for commercial use in 1988, is a widely utilized antifungal drug whose mechanism of action involves inhibition of the activity of 14-α lanosterol demethylase. Its safety and effectiveness have established it as one of the most frequently employed antifungal agents. Resistance to azole antifungal drugs is becoming more common. It may be related to a mutation of the gene encoding the enzyme. To address this issue, molecules with modifications in three main regions of fluconazole, namely the hydroxyl group, the aromatic ring, and the 1,2,4-triazole rings, have been synthesized in an attempt to create more potent antifungal drugs. These modifications aim at enhancing the effectiveness against microorganisms and improving pharmacokinetic parameters and safety profiles of the synthesized compounds. The present review explores the synthesis of fluconazole derivatives, accompanied by insights into the results of biological studies evaluating the therapeutic effects of these compounds.

Candidiasis places a significant burden on human health and can range from common superficial vulvovaginal and oral infections to invasive diseases with high mortality. The most common Candida species implicated in human disease is Candida albicans , but other species like Candida glabrata are emerging. The use of azole antifungals for treatment is limited by increasing rates of resistance. This study explores repositioning bisphosphonates, which are traditionally used for osteoporosis, as antifungal synergists that can improve and revitalize the use of azoles. Risedronate, alendronate, and zoledronate (ZOL) were tested against isolates from six different species of Candida , and ZOL produced moderate antifungal activity and strong synergy with azoles like fluconazole (FLC), particularly in C. glabrata . FLC:ZOL combinations had increased fungicidal and antibiofilm activity compared to either drug alone, and the combination prevented the development of antifungal resistance. Mechanistic investigations demonstrated that the synergy was mediated by the depletion of squalene, resulting in the inhibition of ergosterol biosynthesis and a compromised membrane structure. In C. glabrata , synergy compromised the function of membrane-bound multidrug transporters and caused an accumulation of reactive oxygen species, which may account for its acute sensitivity to FLC:ZOL. The efficacy of FLC:ZOL in vivo was confirmed in a Galleria mellonella infection model, where combinations improved the survival of larvae infected with C. albicans and C. glabrata to a greater extent than monotherapy with FLC or ZOL, and at reduced dosages. These findings demonstrate that bisphosphonates and azoles are a promising new combination therapy for the treatment of topical candidiasis., Importance: Candida is a common and often very serious opportunistic fungal pathogen. Invasive candidiasis is a prevalent cause of nosocomial infections with a high mortality rate, and mucocutaneous infections significantly impact the quality of life of millions of patients a year. These infections pose substantial clinical challenges, particularly as the currently available antifungal treatment options are limited in efficacy and often toxic. Azoles are a mainstay of antifungal therapy and work by targeting the biosynthesis of ergosterol. However, there are rising rates of acquired azole resistance in various Candida species, and some species are considered intrinsically resistant to most azoles. Our research demonstrates the promising therapeutic potential of synergistically enhancing azoles with non-toxic, FDA-approved bisphosphonates. Repurposing bisphosphonates as antifungal synergists can bypass much of the drug development pipeline and accelerate the translation of azole-bisphosphonate combination therapy., Competing Interests: The authors declare no conflict of interest.

Introduction: Since the drug resistance in Candida species is becoming a serious clinical challenge, novel alternative therapeutic options, particularly herbal medicine, have attracted increasing interest. This study aimed to pinpoint the potential antifungal activity of crocin (Cro), the efficacy of the niosomal formulation of Cro (NCro), and the synergistic activity of both formulations in combination with fluconazole (FLC) against susceptible and resistant C. albicans isolates., Material and Methods: NCro was formulated using the heating method. The in vitro antimycotic activity of Cro, NCro, and FLC was evaluated. Checkerboard and isobologram assays evaluated the interaction between both formulations of Cro and FLC. Necrotic and apoptotic effects of different agents were analyzed using the flow cytometry method. In silico study was performed to examine the interactions between Lanosterol 14 alpha-demethylase and Cro as a part of our screening compounds with antifungal properties., Results: NCro exhibited high entrapment efficiency up to 99.73 ± 0.54, and the mean size at 5.224 ± 0.618 µm (mean ± SD, n = 3). Both formulations of Cro were shown to display good anticandidal activity against isolates. The synergistic effect of the NCro in combination with FLC is comparable to Cro (P-value =0.03). Apoptotic indicators confirmed that tested compounds caused cell death in isolates. The docking study indicated that Cro has interactivity with the protein residue of 14α-demethylase., Conclusion: The results showed a remarkable antifungal effect by NCro combined with FLC. Natural compounds, particularly nano-sized carrier systems, can act as an effective therapeutic option for further optimizing fungal infection treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 SFMM. Published by Elsevier Masson SAS. All rights reserved.)

Botrytis cinerea is a severe threat in agriculture, as it can infect over 200 different crop species with gray mold affecting food yields and quality. The conventional treatment using fungicides lead to emerging resistance over the past decades. Here, we introduce Photodynamic Inactivation (PDI) as a strategy to combat B. cinerea infections, independent of fungicide resistance. PDI uses photoactive compounds, which upon illumination create reactive oxygen species toxic for killing target organisms. This study focuses on different formulations of sodium-magnesium-chlorophyllin (Chl, food additive E140) as photoactive compound in combination with EDTA disodium salt dihydrate (Na 2 EDTA) as cell-wall permeabilizer and a surfactant. In an in vitro experiment, three different photosensitizers (PS) with varying Chl and Na 2 EDTA concentrations were tested against five B. cinerea strains with different resistance mechanisms. We showed that all B. cinerea mycelial spheres of all tested strains were eradicated with concentrations as low as 224 µM Chl and 3.076 mM Na 2 EDTA (LED illumination with main wavelength of 395 nm, radiant exposure 106 J cm -2 ). To further test PDI as a Botrytis treatment strategy in agriculture a greenhouse trial was performed on B. cinerea infected bell pepper plants (Capsicum annum L). Two different rates (560 or 1120 g Ha -1 ) of PS formulation (0.204 M Chl and 1.279 M Na 2 EDTA) and a combination of PS formulation with 0.05% of the surfactant BRIJ L4 (560 g Ha -1 ) were applied weekly for 4 weeks by spray application. Foliar lesions, percentage of leaves affected, percentage of leaf area diseased and AUDPC were significantly reduced, while percentage of marketable plants were increased by all treatments compared to a water treated control, however, did not statistically differ from each other. No phytotoxicity was observed in any treatment. These results add to the proposition of employing PDI with the naturally sourced PS Chl in agricultural settings aimed at controlling B. cinerea disease. This approach seems to be effective regardless of the evolving resistance mechanisms observed in response to conventional antifungal treatments., (© 2024. The Author(s).)

The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene., Competing Interests: he authors have declared that no competing interests exist., (Copyright: © 2024 Manzoor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance., (© 2024. The Author(s).)

Thirty-one novel albaconazole derivatives were designed and synthesized based on our previous work. All compounds exhibited potent in vitro antifungal activities against seven pathogenic fungi. Among them, tetrazole compound D2 was the most potent antifungal with MIC values of <0.008, <0.008, and 2 μg/mL against Candida albicans , Cryptococcus neoformans , and Aspergillus fumigatus , respectively, the three most common and critical priority pathogenic fungi. In addition, compound D2 also exhibited potent activity against fluconazole-resistant C. auris isolates. Notably, compound D2 showed a lower inhibitory activity in vitro against human CYP450 enzymes as well as a lower inhibitory effect on the hERG K + channel, indicating a low risk of drug-drug interactions and QT prolongation. Moreover, with improved pharmacokinetic profiles, compound D2 showed better in vivo efficacy than albaconazole at reducing fungal burden and extending the survival of C. albicans -infected mice. Taken together, compound D2 will be further investigated as a promising candidate.

Fungal infections are emerging as major health challenges in recent years. The development of resistance against existing antifungal agents needs urgent attention and action. The limited classes of antifungal drugs available, their tendency to cause adverse effects, lack of effectiveness, etc., are the major limitations of current therapy. Thus, there is a pressing demand for new antifungal drug classes to cope with the present circumstances. Glucose is the key source of energy for all organisms, including fungi. Glucose plays a crucial role as a source of carbon and energy for processes like virulence, growth, invasion, biofilm formation, and resistance development. The glucose transport and sensing mechanisms are well developed in these organisms as an important strategy to sustain survival. Modulating these transport or sensor mechanisms may serve as an important strategy to inhibit fungal growth. Moreover, the structural difference between human and fungal glucose transporters makes them more appealing as drug targets. Limited literature is available for fungal glucose entry mechanisms. This review provides a comprehensive account of sugar transport mechanisms in common fungal pathogens., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Dermatophyte infections (a.k.a. ringworm, tinea) affect an estimated 20%-25% of the world's population. In North America, most dermatophytoses are caused by Trichophyton rubrum or Trichophyton mentagrophytes species complexes. Severe and antifungal-resistant dermatophytoses are a growing global public health problem. A new species of the T. mentagrophytes species complex, Trichophyton indotineae , has recently emerged and is notable for the severe infections it causes, its propensity for antifungal resistance, and its global spread. In this issue of the Journal of Clinical Microbiology , C. F. Cañete-Gibas, J. Mele, H. P. Patterson, et al. (J Clin Microbiol 61:e00562-23, 2023, https://doi.org/10.1128/JCM.00562-23) summarize the results of speciation and AFST performed on North American dermatophyte isolates received at a fungal diagnostic reference laboratory. Within their collection, 18.6% of isolates were resistant to terbinafine (a first-line oral antifungal for dermatophytoses), and similar proportions of T. rubrum and T. indotineae demonstrated terbinafine resistance. The authors also found that T. indotineae has been present in North America since at least 2017. These findings highlight the importance of increased surveillance efforts to monitor trends in severe and antifungal-resistant dermatophytoses and the need for antifungal stewardship efforts, the success of which is contingent upon improving laboratory capacity for dermatophyte speciation and AFST., Competing Interests: The authors declare no conflict of interest.

The membrane-integrated synthase FKS is involved in the biosynthesis of β-1,3-glucan, the core component of the fungal cell wall 1,2 . FKS is the target of widely prescribed antifungal drugs, including echinocandin and ibrexafungerp 3,4 . Unfortunately, the mechanism of action of FKS remains enigmatic and this has hampered development of more effective medicines targeting the enzyme. Here we present the cryo-electron microscopy structures of Saccharomyces cerevisiae FKS1 and the echinocandin-resistant mutant FKS1(S643P). These structures reveal the active site of the enzyme at the membrane-cytoplasm interface and a glucan translocation path spanning the membrane bilayer. Multiple bound lipids and notable membrane distortions are observed in the FKS1 structures, suggesting active FKS1-membrane interactions. Echinocandin-resistant mutations are clustered at a region near TM5-6 and TM8 of FKS1. The structure of FKS1(S643P) reveals altered lipid arrangements in this region, suggesting a drug-resistant mechanism of the mutant enzyme. The structures, the catalytic mechanism and the molecular insights into drug-resistant mutations of FKS1 revealed in this study advance the mechanistic understanding of fungal β-1,3-glucan biosynthesis and establish a foundation for developing new antifungal drugs by targeting FKS., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

What Is This Summary About?: Fungi are types of microbes that include molds and yeasts. Fungal infections can make people ill and can even cause death, especially in older people. They can be treated using antifungal drugs, but some fungi are drug resistant. This means the drug cannot kill the fungi. This is a summary based on a study that looked at fungal samples to find out more about antifungal drug resistance in adults younger than 65 compared with adults aged 65 and older., What Were the Results?: The study found that one type of drug-resistant fungus, called Candida parapsilosis, was more common in older people than in younger people. Another type, called Aspergillus fumigatus, was more common in younger people than in older people. We also found genetic changes in drug-resistant fungi. These changes could explain why the drugs did not work., What Do the Results Mean?: We hope that the findings from this study can help scientists create new treatments for drug-resistant fungal infections.

In the present study, in vitro and in vivo interactions of TOR inhibitor AZD8055 and azoles, including itraconazole, voriconazole, posaconazole and fluconazole, against a variety of pathogenic fungi were investigated. A total of 69 isolates were studied via broth microdilution checkerboard technique, including 23 isolates of Aspergillus spp., 20 isolates of Candida spp., 9 isolates of Cryptococcus neoformans complex, and 17 isolates of Exophiala dermatitidis . The results revealed that AZD8055 individually did not exert any significant antifungal activity. However, synergistic effects between AZD8055 and itraconazole, voriconazole or posaconazole were observed in 23 (33%), 13 (19%) and 57 (83%) isolates, respectively, including azole-resistant A. fumigatus strains and Candida spp., potentiating the efficacy of azoles. The combination effect of AZD8055 and fluconazole was investigated against non-auris Candida spp. and C. neoformans complex. Synergism between AZD8055 and fluconazole was observed in six strains (60%) of Candida spp., resulting in reversion of fluconazole resistance. Synergistic combinations resulted in 4-fold to 256-fold reduction of effective MICs of AZD8055 and azoles. No antagonism was observed. In vivo effects of AZD8055-azole combinations were evaluated by survival assay in Galleria mellonella model infected with A. fumigatus strain AF002, E. dermatitidis strain BMU00038, C. auris strain 383, C. albicans strain R15, and C. neoformans complex strain Z2. AZD8055 acted synergistically with azoles and significantly increased larvae survival ( P < 0.05). In summary, the results suggested that AZD8055 combined with azoles may help to enhance the antifungal susceptibilities of azoles against pathogenic fungi and had the potential to overcome azole resistance issues. IMPORTANCE Limited options of antifungals and the emergence of drug resistance in fungal pathogens has been a multifaceted clinical challenge. Combination therapy represents a valuable alternative to antifungal monotherapy. The target of rapamycin (TOR), a conserved serine/threonine kinase from yeast to humans, participates in a signaling pathway that governs cell growth and proliferation in response to nutrient availability, growth factors, and environmental stimuli. AZD8055 is an orally bioavailable, potent, and selective TOR kinase inhibitor that binds to the ATP binding cleft of TOR kinase and inhibits both TORC1 and TORC2. Synergism between AZD8055 and azoles suggested that the concomitant application of AZD8055 and azoles may help to enhance azole therapeutic efficacy and impede azole resistance. TOR inhibitor with fungal specific target is promising to be served as combination regimen with azoles.

With the widespread use of azole antifungals in the clinic, the drug resistance has been emerging continuously. In this work, we focus on boron trifluoride etherate catalyzed condensation of indole and salicylaldehydes to form bis(indolyl)methanes (BIMs) in high yields, and in vitro antifungal activity against Candida albicans were evaluated. The results showed that most phenol-derived BIMs combined with fluconazole (FLC) exhibited good antifungal activity against sensitive and drug-resistant C. albicans. Further mechanism study demonstrated that BI-10 combined with FLC could inhibit hyphal growth, result in ROS accumulation, and decrease mitochondrial membrane potential (MMP) as well as altering membrane permeability., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Candida species are the most common fungal pathogens infecting humans and can cause severe illnesses in immunocompromised individuals. The increased resistance of Candida to traditional antifungal drugs represents a great challenge in clinical settings. Therefore, novel approaches to overcome antifungal resistance are desired. Here, we investigated the use of an antimicrobial peptide WMR against Candida albicans and non- albicans Candida species in vitro and in vivo. Results showed a WMR antifungal activity on all Candida planktonic cells at concentrations between 25 μM to >50 μM and exhibited activity at sub-MIC concentrations to inhibit biofilm formation and eradicate mature biofilm. Furthermore, in vitro antifungal effects of WMR were confirmed in vivo as demonstrated by a prolonged survival rate of larvae infected by Candida species when the peptide was administered before or after infection. Additional experiments to unravel the antifungal mechanism were performed on C. albicans and C. parapsilosis . The time-killing curves showed their antifungal activity, which was further confirmed by the induced intracellular and mitochondrial reactive oxygen species accumulation; WMR significantly suppressed drug efflux, down-regulating the drug transporter encoding genes CDR1 . Moreover, the ability of WMR to penetrate within the cells was demonstrated by confocal laser scanning microscopy. These findings provide novel insights for the antifungal mechanism of WMR against Candida albicans and non- albicans , providing fascinating scenarios for the identification of new potential antifungal targets.

Resistance to antifungal therapy of Candida albicans and non- albicans Candida strains, frequently associated with oral candidosis, is on the rise. In this context, host-defense peptides have emerged as new promising candidates to overcome antifungal resistance. Thus, the aim of this study was to assess the effectiveness against Candida species of different Catestatin-derived peptides, as well as the combined effect with serum albumin. Among Catestatin-derived peptides, the most active against sensitive and resistant strains of C . albicans , C. tropicalis and C. glabrata was the D -isomer of Cateslytin ( D -bCtl) whereas the efficiency of the L -isomer ( L -bCtl) significantly decreases against C. glabrata strains. Images obtained by transmission electron microscopy clearly demonstrated fungal membrane lysis and the leakage of the intracellular material induced by the L -bCtl and D -bCtl peptides. The possible synergistic effect of albumin on Catestatin-derived peptides activity was investigated too. Our finding showed that bovine serum albumin (BSA) when combined with the L - isomer of Catestatin ( L -bCts) had a synergistic effect against Candida albicans especially at low concentrations of BSA; however, no synergistic effect was detected when BSA interacted with L -bCtl, suggesting the importance of the C-terminal end of L -bCts (GPGLQL) for the interaction with BSA. In this context in vitro D -bCtl, as well as the combination of BSA with L -bCts are potential candidates for the development of new antifungal drugs for the treatment of oral candidosis due to Candida and non- Candida albicans , without detrimental side effects.

Fructose-1,6-bisphosphate aldolase (FBA) represents an attractive new antifungal target. Here, we employed a structure-based optimization strategy to discover a novel covalent binding site (C292 site) and the first-in-class covalent allosteric inhibitors of FBA from Candida albicans (CaFBA). Site-directed mutagenesis, liquid chromatography-mass spectrometry, and the crystallographic structures of APO-CaFBA, CaFBA- G3P , and C157S- 2a4 revealed that S268 is an essential pharmacophore for the catalytic activity of CaFBA, and L288 is an allosteric regulation switch for CaFBA. Furthermore, most of the CaFBA covalent inhibitors exhibited good inhibitory activity against azole-resistant C. albicans , and compound 2a11 can inhibit the growth of azole-resistant strains 103 with the MIC 80 of 1 μg/mL. Collectively, this work identifies a new covalent allosteric site of CaFBA and discovers the first generation of covalent inhibitors for fungal FBA with potent inhibitory activity against resistant fungi, establishing a structural foundation and providing a promising strategy for the design of potent antifungal drugs.

Antifungal susceptibility testing is an essential tool for guiding antifungal therapy. Reference methods are complex and usually only available in specialised laboratories. We have designed an expanded agar-based screening method for the detection of azole-resistant Aspergillus fumigatus isolates. Normally, identification of resistance mechanisms is obtained only after sequencing the cyp51A gene and promoter. However, our screening method provides azole resistance detection and presumptive resistance mechanisms identification. A previous agar-based method consisting of four wells containing voriconazole, itraconazole, posaconazole and a growth control, detected azole resistance to clinical azoles. Here, we have modified the concentrations of voriconazole and posaconazole to adapt to the updated EUCAST breakpoints against A. fumigatus. We have also expanded the method to include environmental azoles to assess azole resistance and the azole resistance mechanism involved. We used a collection of A. fumigatus including 54 azole-resistant isolates with Cyp51A modifications (G54, M220, G448S, TR 53 , TR 34 /L98H, TR 46 /Y121F/T289A, TR 34 /L98H/S297T/F495I), and 50 azole susceptible isolates with wild-type Cyp51A. The screening method detects azole-resistant A. fumigatus isolates when there is growth in any of the azole-containing wells after 48h. The growth pattern in the seven azoles tested helps determine the underlying azole resistance mechanism. This approach is designed for surveillance screening of A. fumigatus azole-resistant isolates and can be useful for the clinical management of patients prior to antifungal susceptibility testing confirmation., (© 2021 Wiley-VCH GmbH.)

The aim of this study was to uncover the molecular mechanism through which fungicide resistance develops in Podosphaera xanthii, a fungi that causes powdery mildew in hull-less pumpkin. Treatments of inoculated P. xanthii were carried out on leaves of hull-less pumpkin and subsequently treated with kinds of triazole fungicide for seven generations. Resistant strains of P. xanthii thus obtained were evaluated for their resistance levels. The resistance levels of the fungi to four fungicides of were high except that of the propiconazole-resistant strain, which showed moderate resistance. The F7 generations of five resistant strains thus obtained were cultured continuously for five generations without fungicide induction, and their resistance level were found to be relatively stable. The DNA of the sensitive strain and the five kinds of resistant strains were extracted by the sodium dodecyl sulfate (SDS) method and its internal transcribed spacer (ITS) region was amplified by using ITS1/ITS4 primer and specific primer F/R and they were sequenced respectively. The DNA sequence comparison of resistant and sensitive strains showed that the base pairs of tebuconazole-resistant strains and flusilazole-resistant strains were mutated, with mutation rates of 4.8% and 1.6%, respectively. The base pairs of the other three resistant strains did not change., Competing Interests: The authors have declared that no competing interests exist.

SYAUP-CN-26 (1 S , 2 R -((3-bromophenethyl)amino)- N -(4-chloro-2-trifluoromethylphenyl) cyclohexane-1-sulfonamide) is a novel sulfonamide compound with excellent activity against Botrytis cinerea . The present study sought to explore the mutant of B. cinerea resistant to SYAUP-CN-26 using SYAUP-CN-26 plates. Moreover, the cell membrane functions of B. cinerea , histidine kinase activity, relative conductivity, triglyceride, and cell membrane structure were determined, and the target gene histidine kinase Bos1 (AF396827.2) of procymidone was amplified and sequenced. The results showed that compared to the sensitive strain, the cell membrane permeability, triglyceride, and histidine kinase activity of the resistant strain showed significant changes. The relative conductivity of the sensitive strain increased by 6.95% and 9.61%, while the relative conductivity of the resistant strain increased by 0.23% and 1.76% with 26.785 µg/mL (EC 95 ) and 79.754 µg/mL (MIC) of SYAUP-CN-26 treatment. The triglyceride inhibition rate of the resistant strain was 23.49% and 37.80%, which was 0.23% and 1.76% higher than the sensitive strain. Compared to the sensitive strain, the histidine kinase activity of the resistant strain was increased by 23.07% and 35.61%, respectively. SYAUP-CN-26 significantly damaged the cell membrane structure of the sensitive strain. The sequencing of the Bos1 gene of the sensitive and resistant strains indicated that SYAUP-CN-26 resistance was associated with a single point mutation (P348L) in the Bos1 gene. Therefore, it was inferred that the mutant of B. cinerea resistant to SYAUP-CN-26 might be regulated by the Bos1 gene. This study will provide a theoretical basis for further research and development of sulfonamide compounds for B. cinerea and new agents for the prevention and control of resistant B. cinerea .

5-phenylthiophene derivatives exhibited excellent antifungal activity against Candida albicans, Candida tropicalis and Cryptococcus neoformans. However, optimal compound 7 was inactive against Aspergillus fumigatus and unstable in human liver microsomes in vitro with a half-life of 18.6 min. To discover antifungal agents with a broad spectrum and improve the metabolic properties of the compounds, the scaffold hopping strategy was adopted and a series of 4-phenyl-4,5-dihydrooxazole derivatives were designed and synthesized. It was especially encouraging that compound 22a displayed significant antifungal activities against eight susceptible strains and seven FLC-resistant strains. Furthermore, the potent compound 22a could prevent the formation of fungalbiofilms and displayed satisfactory fungicidal activity. In addition, the metabolic stability of compound 22a was improved significantly, with the half-life of 70.5 min. Compound 22a was almost nontoxic to mammalian A549, MCF-7, HepG2, and 293T cells. Moreover, pharmacokinetic studies in SD rats showed that compound 22a exhibited pharmacokinetic properties with a bioavailability of 15.22% and a half-life of 4.44 h, indicating that compound 22a is worthy of further study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Clinical treatment of candidiasis has suffered from increasingly severe drug resistance and limited efficacy. Thus, novel strategies to deal with drug resistance are highly desired to develop effective therapeutic agents. Herein, dual inhibition of heat shock protein 90 (Hsp90) and histone deacetylase (HDAC) was validated as a new strategy to potentiate efficacy of fluconazole against resistant Candida albicans infections. The first generation of Hsp90/HDAC dual inhibitors were designed as synergistic enhancers to treat azoles-resistant candidiasis. In particular, compound J5 exhibited fungal-selective inhibitory effects on Hsp90 and HDACs, leading to low toxicity and excellent in vitro (FICI = 0.266) and in vivo synergistic antifungal potency to treat fluconazole resistant candidiasis. Antifungal-mechanistic investigation revealed that compound J5 suppressed important virulence factors and down-regulated expression of resistance-associated genes. Therefore, Hsp90/HDAC dual inhibitors represent a new strategy for the development of novel antifungal therapeutics to combat azole-resistant candidiasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Vulvovaginal candidiasis (VVC) is the second most common vaginal infection that affects women of reproductive age. Its increased occurrence and associated treatment cost coupled to the rise in resistance of the causative pathogen to current antifungal therapies has necessitated the need for the discovery and development of novel effective antifungal agents for the treatment of the disease. We report in this study the anti-Candida albicans activity of Solanum torvum 70% ethanol fruit extract (STF), fractions and some isolated compounds against four (4) fluconazole-resistant strains of C. albicans. We further report on the effect of the isolated compounds on the antifungal activity of fluconazole and voriconazole in the resistant isolates as well as their inhibitory effect on C. albicans biofilm formation. STF was fractionated using n-hexane, chloroform (CHCl3) and ethyl acetate (EtOAc) to obtain four respective major fractions, which were then evaluated for anti-C. albicans activity using the microbroth dilution method. The whole extract and fractions recorded MICs that ranged from 0.25 to 16.00 mg/mL. From the most active fraction, STF- CHCl3 (MIC = 0.25-1.00 mg/mL), four (4) known compounds were isolated as Betulinic acid, 3-oxo-friedelan-20α-oic acid, Sitosterol-3-β-D-glucopyranoside and Oleanolic acid. The compounds demonstrated considerably higher antifungal activity (0.016 to 0.512 mg/mL) than the extract and fractions and caused a concentration-dependent anti-biofilm formation activity. They also increased the sensitivity of the C. albicans isolates to fluconazole. This is the first report of 3-oxo-friedelan-20α-oic acid in the plant as well as the first report of betulinic acid, sitosterol-3-β-D-glucopyranoside and oleanolic acid from the fruits of S. torvum. The present study has demonstrated the anti-C. albicans activity of the constituents of S. torvum ethanol fruit extract and also shown that the constituents possess anti-biofilm formation and resistance modulatory activities against fluconazole-resistant clinical C. albicans isolates., Competing Interests: The authors have declared that no competing interests exist.

Resistance to fluconazole is one of clinical characteristics most frequently challenging the treatment of invasive Candida auris infections, and is observed among >90% of all characterized clinical isolates. In this work, the native C. auris ERG11 allele in a previously characterized fluconazole-susceptible clinical isolate was replaced with the ERG11 alleles from three highly fluconazole-resistant clinical isolates (MIC ≥256 mg/L), encoding the amino acid substitutions VF125AL, Y132F, and K143R, using Cas9-ribonucleoprotein (RNP) mediated transformation system. Reciprocally, the ERG11 WT allele from the same fluconazole-susceptible clinical isolate, lacking any resistance-associated mutation, was introduced into a previously characterized fluconazole-resistant clinical isolate, replacing the native ERG11 K143R allele, using the same methods. The resulting collection of strains was subjected to comprehensive triazole susceptibility testing, and the direct impact each of these clinically-derived ERG11 mutations on triazole MIC was determined. Introduction of each of the three mutant ERG11 alleles was observed to increase fluconazole and voriconazole MIC by 8- to 16-fold. The MIC for the other clinically available triazoles were not significantly impacted by any ERG11 mutation. In the fluconazole-resistant clinical isolate background, correction of the K143R encoding mutation led to a similar 16-fold decrease in fluconazole MIC, and 8-fold decrease in voriconazole MIC, while the MIC of other triazoles were minimally changed. Taken together, these findings demonstrate that mutations in C. auris ERG11 significantly contribute to fluconazole and voriconazole resistance, but alone cannot explain the substantially elevated MIC observed among clinical isolates of C. auris. IMPORTANCE Candida auris is an emerging multidrug-resistant and health care-associated pathogen of urgent clinical concern. The triazoles are the most widely prescribed antifungal agents worldwide and are commonly utilized for the treatment of invasive Candida infections. Greater than 90% of all C. auris clinical isolates are observed to be resistant to fluconazole, and nearly all fluconazole-resistant isolates of C. auris are found to have one of three mutations (encoding VF125AL, Y132F, or K143R) in the gene encoding the target of the triazoles, ERG11 . However, the direct contribution of these mutations in ERG11 to fluconazole resistance and the impact these mutations may have the susceptibility of the other triazoles remains unknown. The present study seeks to address this knowledge gap and potentially inform the future application the triazole antifungals for the treatment of infections caused by C. auris.

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC 50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance., (© 2021. The Author(s).)

Diabetic foot ulcer (DFU) is a devastating complication, affecting around 15% of diabetic patients and representing a leading cause of non-traumatic amputations. Notably, the risk of mixed bacterial-fungal infection is elevated and highly associated with wound necrosis and poor clinical outcomes. However, it is often underestimated in the literature. Therefore, polymicrobial infection control must be considered for effective management of DFU. It is noteworthy that antimicrobial resistance is constantly rising overtime, therefore increasing the need for new alternatives to antibiotics and antifungals. Antimicrobial peptides (AMPs) are endogenous peptides that are naturally abundant in several organisms, such as bacteria, amphibians and mammals, particularly in the skin. These molecules have shown broad-spectrum antimicrobial activity and some of them even have wound-healing activity, establishing themselves as ideal candidates for treating multi-kingdom infected wounds. Furthermore, the role of AMPs with antifungal activity in wound management is poorly described and deserves further investigation in association with antibacterial agents, such as antibiotics and AMPs with antibacterial activity, or alternatively the application of broad-spectrum antimicrobial agents that target both aerobic and anaerobic bacteria, as well as fungi. Accordingly, the aim of this review is to unravel the molecular mechanisms by which AMPs achieve their dual antimicrobial and wound-healing properties, and to discuss how these are currently being applied as promising therapies against polymicrobial-infected chronic wounds such as DFUs.

Candida auris is an emerging healthcare-associated pathogen of global concern. Recent reports have identified C. auris isolates that grow in cellular aggregates or filaments, often without a clear genetic explanation. To investigate the regulation of C. auris morphogenesis, we applied an Agrobacterium-mediated transformation system to all four C. auris clades. We identified aggregating mutants associated with disruption of chitin regulation, while disruption of ELM1 produced a polarized, filamentous growth morphology. We developed a transiently expressed Cas9 and sgRNA system for C. auris that significantly increased targeted transformation efficiency across the four C. auris clades. Using this system, we confirmed the roles of C. auris morphogenesis regulators. Morphogenic mutants showed dysregulated chitinase expression, attenuated virulence, and altered antifungal susceptibility. Our findings provide insights into the genetic regulation of aggregating and filamentous morphogenesis in C. auris. Furthermore, the genetic tools described here will allow for efficient manipulation of the C. auris genome., (© 2021. The Author(s).)

Fungal infections are underestimated threats that affect over 1 billion people, and Candida spp., Cryptococcus spp., and Aspergillus spp. are the 3 most fatal fungi. The treatment of these infections is performed with a limited arsenal of antifungal drugs, and the class of the azoles is the most used. Although these drugs present low toxicity for the host, there is an emergence of therapeutic failure due to azole resistance. Drug resistance normally develops in patients undergoing azole long-term therapy, when the fungus in contact with the drug can adapt and survive. Conversely, several reports have been showing that resistant isolates are also recovered from patients with no prior history of azole therapy, suggesting that other routes might be driving antifungal resistance. Intriguingly, antifungal resistance also happens in the environment since resistant strains have been isolated from plant materials, soil, decomposing matter, and compost, where important human fungal pathogens live. As the resistant fungi can be isolated from the environment, in places where agrochemicals are extensively used in agriculture and wood industry, the hypothesis that fungicides could be driving and selecting resistance mechanism in nature, before the contact of the fungus with the host, has gained more attention. The effects of fungicide exposure on fungal resistance have been extensively studied in Aspergillus fumigatus and less investigated in other human fungal pathogens. Here, we discuss not only classic and recent studies showing that environmental azole exposure selects cross-resistance to medical azoles in A. fumigatus, but also how this phenomenon affects Candida and Cryptococcus, other 2 important human fungal pathogens found in the environment. We also examine data showing that fungicide exposure can select relevant changes in the morphophysiology and virulence of those pathogens, suggesting that its effect goes beyond the cross-resistance., Competing Interests: The authors have declared that no competing interests exist.

Fungal infections pose a significant health burden. In a new study, Candida glabrata isolates resistant to mechanistically distinct antifungals were evolved, unveiling mutations in ERG3 as a novel mechanism by which this pathogen evolves multidrug resistance., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A series of 5-phenylthiophene derivatives with novel structures were designed and synthesized to combat the increasing incidence of susceptible and drug-resistant fungal infections. The antifungal activity of the synthesized compounds was assessed against seven susceptible strains and six fluconazole-resistant strains. It is especially encouraging that compounds 17b and 17f displayed significant antifungal activities against all tested strains. Furthermore, the potent compounds 17b and 17f could prevent the formation of fungi biofilms and 17f displayed satisfactory fungicidal activity. Preliminary mechanistic studies showed that the potent antifungal activity of compound 17f stemmed from inhibition of C. albicans CYP51. In addition, Compounds 17b and 17f were almost nontoxic to mammalian A549, MCF-7, and THLE-2 cells. These results strongly suggested that compounds 17b and 17f are promising as novel antifungal drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

In the past decade, researchers have focused on the emergence of drug resistance in fungal pathogens such as Candida albicans, also considered as pathobionts that occur harmlessly in the human body but could potentially be triggered to cause diseases. The increasing rate of antifungal resistance in commensal gut fungi is alarming and should be further investigated. Here, we report seven novel MLST (Multi Locus Sequence Typing) genotypes of multi-drug resistant C. albicans isolates obtained from participants of a community study in Segamat, a district in the state of Johor, Malaysia. A total of eight C. albicans were isolated from four individuals, which were found to express high resistance against fluconazole, itraconazole, voriconazole and 5-fluorocytosine antifungals. MLST was performed to assess the clonal relatedness of these drug resistant isolates among themselves and against other strains isolated from other geographical regions. The novel MLST C. albicans sequence types suggest significant genetic changes compared to previous genotypes., (© 2021. Sociedade Brasileira de Microbiologia.)

Purpose: The resistance of C. albicans to traditional antifungal drugs brings a great challenge to clinical treatment. To overcome the resistance, developing antifungal agent sensitizers has attracted considerable attention. This study aimed to determine the anti- Candida activity of BEH alone or BEH-FLC combination and to explore the underlying mechanisms., Materials and Methods: In vitro antifungal effects were performed by broth microdilution assay and XTT reduction assay. Infected Galleria mellonella larvae model was used to determine the antifungal effects in vivo. Probes Fluo-3/AM, FITC-VAD-FMK and rhodamine 6G were used to study the influence of BEH and FLC on intracellular calcium concentration, metacaspase activity and drug efflux of C. albicans ., Results: BEH alone exhibited obvious antifungal activities against C. albicans . BEH plus FLC not only showed synergistic effects against planktonic cells and preformed biofilms within 8 h but also enhanced the antifungal activity in infected G. mellonella larvae. Mechanistic studies indicated that antifungal effects of drugs might be associated with the increasement of calcium concentration, activation of metacaspase activity to reduce virulence and anti-biofilms, but were not related to drug efflux., Conclusion: BEH alone or combined with FLC displayed potent antifungal activity both in vitro and in vivo, and the underlying mechanisms were related to reduced virulence factors., Competing Interests: The authors report a patent ZL202010782426.2 licensed to Shujuan Sun; Xueqi Chen; Chunyan Li; Haiying Yan; Hongyao Ren. The authors report no other conflicts of interest in this work., (© 2021 Chen et al.)

Cryptococcosis by Cryptococcus gattii is endemic in Colombia, affecting mostly immunocompetent hosts. Since antifungal susceptibility differs between molecular types of cryptococcal isolates, as reported elsewhere, the aim of this study was to determine if 42 Colombian clinical isolates, VGI, VGII and VGIII, differ in the susceptibility to commonly used antifungals, using Sensititre plates. Among the molecular types, six non-wild type isolates to fluconazole, voriconazole, and 5-flucytosine, were identified. Besides, VGI and VGII were less susceptible to 5-flucytosine and azoles, respectively, than other molecular types. These findings support the applicability of practicing susceptibility testing, which could better guide treatment in cryptococcosis., Lay Summary: Cryptococcosis gattii affects immunocompetent people. For a correct treatment, antifungal susceptibility testing is essential. This study shows differences in the susceptibility to commonly used antimycotics among genotypes of Colombian clinical C. gattii isolates, some of which are non-wild-type., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Mortality rates due to Cryptococcus neoformans var. grubii fungemia remain significant despite treatment with antifungal drugs. The predictive function of antifungal susceptibility and its correlation with treatment outcome remains controversial. A retrospective study was conducted from January 1, 2009, to December 31, 2016, on 85 patients with C. neoformans var. grubii fungemia confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Antifungal drug susceptibility was determined using the YeastONE™ colorimetric broth microdilution method coupled with Vizion™ System following the Clinical and Laboratory Standards Institute guidelines. Six antifungal agents-amphotericin B, fluconazole, flucytosine, itraconazole, posaconazole, and voriconazole-were tested. The patients' demographic data and clinical information were abstracted for further analyses. Antifungal regimens consisting of amphotericin B with or without fluconazole or flucytosine were administered for induction treatment of these patients, followed with intravenous or oral fluconazole for maintenance therapy. Clinical outcomes were defined by 14- and 30-day mortality rates. Risk factors associated with outcomes were fitted in a logistic regression model by univariate or multivariate method. Eighty-five patients with C. neoformans var. grubii fungemia were enrolled in the study. The Sequential Organ Failure Assessment Score, Glasgow Coma Scale, Charlson comorbidity score, and adequate duration of therapy for amphotericin B were predictors for mortality in univariate analysis. Antifungal susceptibility testing with YeastONE™ does not predict clinical outcomes of C. neoformans var. grubii fungemia. Greater disease severity, high comorbidities, poor consciousness level, and inappropriate treatment were associated with increased mortality in cryptococcemia cases., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Objective: This study intends to analyze the data of fungemia in a large tertiary hospital from 2010 to 2019, and is aimed at understanding its epidemic characteristics and drug resistance., Methods: The "Hospital Infection Real-Time Monitoring System" was used to retrieve the case information of patients who were hospitalized for more than 48 hours from 2010 to 2019. The questionnaire was designed to collect patients' basic information, infection situation, drug resistance, and other related information. Statistical software was used for analysis., Results: The fungi detection rate was in the range of 0.19%~0.75% in ten years, the average rate was 0.29%, and the rate 0.2%~0.3% since 2013, which was lower than that from 2010 to 2012. Non- Candida albicans was the main fungus, accounting for 62.50%. The drug resistance of non- C. albicans was higher than that of C. albicans , among which C. glabrata had the highest resistance rate. Data analysis showed that the patients with more serious basic diseases, combined with infection of other sites, surgery, long hospital stay, combination of antibiotics, and invasive catheterization, were more likely to occur fungemia., Conclusion: We should pay more attention to the patients with high-risk factors of fungemia and focus on the drug resistance of non- C. albicans , choose the right antifungal drugs, so as to improve the level of diagnosis and treatment., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2021 Yanling Bai et al.)

The high morbidity and mortality of cryptococcal meningitis is due to the limited range of therapeutic options: only three classes of antifungal drugs are available (polyenes [amphotericin B], azoles [fluconazole], and pyrimidine analogues [flucytosine]). Fluconazole is the most widely used antifungal drug in sub-Saharan Africa, where cryptococcal meningitis is a major cause of death in patients infected with HIV. In this study, we found that exposure to fluconazole, even for short times (48 h) at subinhibitory concentrations, drove rapid adaptation of Cryptococcus neoformans serotype A strain H99 via the acquisition of different aneuploid chromosomes. These aneuploidies conferred heteroresistance to fluconazole. Importantly, most of the adaptors were cross-tolerant to flucytosine. Some of the aneuploid adaptors were not heteroresistant to fluconazole but were tolerant to amphotericin B. Thus, exposure to one antifungal drug class can promote adaptation to two antifungal drug classes, highlighting the plasticity of the C. neoformans genome and raising concerns about the rapid reduction in the range of treatment options for cryptococcal infections. IMPORTANCE Cryptococcosis is a globally distributed invasive fungal infection caused by infections with Cryptococcus neoformans or Cryptococcus gattii. Only three classes of therapeutic drugs are clinically available for treating cryptococcosis: polyenes (amphotericin B), azoles (fluconazole), and pyrimidine analogues (flucytosine). Fluconazole is the primary drug available in resource-limited countries. Aneuploidy is a genomic state due to the gain or loss of chromosomes. We found that C. neoformans rapidly adapted to fluconazole by acquiring diverse aneuploidies and that specific aneuploidies enabled improved growth of isolates susceptible (tolerance) to amphotericin B and/or cross-tolerance to both fluconazole and flucytosine. Therefore, aneuploidy is an underlying mechanism of drug tolerance that not only arises rapidly during growth in fluconazole but can also confer tolerance to other antifungal drugs without prior exposure to those drugs. Resistant isolates have high MICs, and all cells grow similarly in medium with the drug, while tolerant isolates test as susceptible and grow slowly at drug concentrations above the MIC.

The fungal pathogen Candida albicans can form biofilms that protect it from drugs and the immune system. The biofilm cells release extracellular vesicles (EVs) that promote extracellular matrix formation and resistance to antifungal drugs. Here, we define functions for numerous EV cargo proteins in biofilm matrix assembly and drug resistance, as well as in fungal cell adhesion and dissemination. We use a machine-learning analysis of cargo proteomic data from mutants with EV production defects to identify 63 candidate gene products for which we construct mutant and complemented strains for study. Among these, 17 mutants display reduced biofilm matrix accumulation and antifungal drug resistance. An additional subset of 8 cargo mutants exhibit defects in adhesion and/or dispersion. Representative cargo proteins are shown to function as EV cargo through the ability of exogenous wild-type EVs to complement mutant phenotypic defects. Most functionally assigned cargo proteins have roles in two or more of the biofilm phases. Our results support that EVs provide community coordination throughout biofilm development in C. albicans., (© 2021. The Author(s).)

Candida is an opportunistic pathogen and a common cause of fungal infections worldwide. Anti-fungal use against Candida infections has resulted in the appearance of resistant strains. The limited choice of anti-fungal therapy means alternative strategies are needed to control fungal infectious diseases. The aim of this study was to evaluate the inhibition of Candida biofilm formation by Hedera rhombea (Korean name: songak) extract. Biofilm formation was assessed using the crystal violet assay which showed a dose dependent reduction in the presence of extract with the biofilm formation inhibitory concentration of C. albicans (IC50 = 12.5μg/ml), C. tropicalis var. tropicalis (IC50 = 25μg/ml), C. parapsilosis var. parapsilosis (IC50 = 6.25μg/ml), C. glabrata (IC50 = 6.25μg/ml), C. tropicalis (IC50 = 12.5μg/ml), and C. parapsilosis (IC50 = 12.5μg/ml) without directly reducing Candida growth. Treatment with 6.25μg/mL of extract increased the antifungal susceptibility to miconazole from 32% decreasing of fungal growth to 98.8% of that based on the fungal growth assay. Treatment of extract dose-dependently reduced the dimorphic transition of Candida based on the dimorphic transition assay and treatment of 3.125μg/mL of extract completely blocked the adherence of Candida to the HaCaT cells. To know the molecular mechanisms of biofilm formation inhibition by extract, qRT-PCR analysis was done, and the extract was found to dose dependently reduce the expression of hyphal-associated genes (ALS3, ECE1, HWP1, PGA50, and PBR1), extracellular matrix genes (GSC1, ZAP1, ADH5, and CSH1), Ras1-cAMP-PKA pathway genes (CYR1, EFG1, and RAS1), Cph2-Tec1 pathway gene (TEC1) and MAP kinases pathway gene (HST7). In this study, Hedera rhombea extract showed inhibition of fungal biofilm formation, activation of antifungal susceptibility, and reduction of infection. These results suggest that fungal biofilm formation is good screen for developing the antifungal adjuvant and Hedera rhombea extract should be a good candidate against biofilm-related fungal infection., Competing Interests: The authors have declared that no competing interests exist.

An unprecedented amount of fungal and fungal-like infections has recently brought about some of the most severe die-offs and extinctions due to fungal drug resistance. Aimed to alleviate the situation, new effort was made to develop novel purinylthiazolylethanone derivatives, which were expected to combat the fungal drug resistance. Some prepared purinylthiazolylethanone derivatives possessed satisfactory inhibitory action towards the tested fungi, among which compound 8c gave a MIC value of 1 μg/mL against C. albicans. The active molecule 8c was able to kill C. albicans with undetectable resistance as well as low hematotoxicity and cytotoxicity. Furthermore, it could hinder the growth of C. albicans biofilm, thus avoiding the occurrence of drug resistance. Mechanism research manifested that purinylthiazolylethanone derivative 8c led to damage of cell wall and membrane disruption, so protein leakage and the cytoplasmic membrane depolarization were observed. On this account, the activity of fungal lactate dehydrogenase was reduced and metabolism was impeded. Meanwhile, the increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) disordered redox equilibrium, giving rise to oxidative damage to fungal cells and fungicidal effect., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Invasive fungal infections remain a challenge due to lack of effective antifungal agents and serious drug resistance. Discovery of antifungal agents with novel antifungal mechanism is important and urgent. Previously, we designed the first CYP51/HDAC dual inhibitors with potent activity against resistant Candida albicans infections. To better understand the antifungal spectrum and synergistic mechanism, herein new CYP51/HDAC dual inhibitors were designed which showed potent in vitro and in vivo antifungal activity against C. neoformans and C. tropicalis infections. Antifungal mechanism studies revealed that the CYP51/HDAC dual inhibitors acted by inhibiting various virulence factors of C. tropicalis and C. neoformans and down-regulating resistance-associated genes. This study highlights the potential of CYP51/HDAC dual inhibitors as a promising strategy for the discovery of novel broad-spectrum antifungal agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Invasive aspergillosis, mainly caused by Aspergillus &nbsp; fumigatus , can lead to severe clinical outcomes in immunocompromised individuals. Antifungal treatment, based on the use of azoles, is crucial to increase survival rates. However, the recent emergence of azole-resistant A. fumigatus isolates is affecting the efficacy of the clinical therapy and lowering the success rate of azole strategies against aspergillosis. Azole resistance mechanisms described to date are mainly associated with mutations in the azole target gene cyp51A that entail structural changes in Cyp51A or overexpression of the gene. However, strains lacking cyp51A modifications but resistant to clinical azoles have recently been detected. Some genes have been proposed as new players in azole resistance. In this study, the gene hmg1 , recently related to azole resistance, and its paralogue hmg2 were studied in a collection of fifteen azole-resistant strains without cyp51A modifications. Both genes encode HMG-CoA reductases and are involved in the ergosterol biosynthesis. Several mutations located in the sterol sensing domain (SSD) of Hmg1 (D242Y, G307D/S, P309L, K319Q, Y368H, F390L and I412T) and Hmg2 (I235S, V303A, I312S, I360F and V397C) were detected. The role of these mutations in conferring azole resistance is discussed in this work.

There are four major classes of antifungals with the predominant mechanism of action being targeting of cell wall or cell membrane. As in other drugs, low solubility of these compounds has led to low bioavailability in target tissues. Enhanced drug dosages have effects such as toxicity, drug-drug interactions, and increased drug resistance by fungi. This article reviews the current state-of-the-art of antifungals, structure, mechanism of action, other usages, and toxic side effects. The emergence of nanoformulations to transport and uniformly release cargo at the target site is a boon in antifungal treatment. The article details research that lead to the development of nanoformulations of antifungals and potential advantages and avoidance of the lacunae characterizing conventional drugs. A range of nanoformulations based on liposomes, polymers are in various stages of research and their potential advantages have been brought out. It could be observed that under similar dosages, test models, and duration, nanoformulations provided enhanced activity, reduced toxicity, higher uptake and higher immunostimulatory effects. In most instances, the mechanism of antifungal activity of nanoformulations was similar to that of regular antifungal. There are possibilities of coupling multiple antifungals on the same nano-platform. Increased activity coupled with multiple mechanisms of action presents for nanoformulations a tremendous opportunity to overcome antifungal resistance. In the years to come, robust methods for the preparation of nanoformulations taking into account the repeatability and reproducibility in action, furthering the studies on nanoformulation toxicity and studies of human models are required before extensive use of nanoformulations as a prescribed drug., (© 2021 Wiley-VCH GmbH.)

Invasive fungal infections mainly affect patients undergoing transplantation, surgery, neoplastic disease, immunocompromised subjects and premature infants, and cause over 1.5 million deaths every year. The most common fungi isolated in invasive diseases are Candida spp., Cryptococcus spp., and Aspergillus spp. and even if four classes of antifungals are available (Azoles, Echinocandins, Polyenes and Pyrimidine analogues), the side effects of drugs and fungal acquired and innate resistance represent the major hurdles to be overcome. Monoclonal antibodies are powerful tools currently used as diagnostic and therapeutic agents in different clinical contexts but not yet developed for the treatment of invasive fungal infections. In this paper we report the development of the first humanized monoclonal antibody specific for β-1,3 glucans, a vital component of several pathogenic fungi. H5K1 has been tested on C. auris, one of the most urgent threats and resulted efficient both alone and in combination with Caspofungin and Amphotericin B showing an enhancement effect. Our results support further preclinical and clinical developments for the use of H5K1 in the treatment of patients in need., (© 2021. The Author(s).)

Candida &nbsp; albicans is the most commonly implicated agent in invasive human fungal infections. The disease could be presented as minimal symptomatic candidemia or can be fulminant sepsis. Candidemia is associated with a high rate of mortality and high healthcare and hospitalization costs. The surveillance programs have reported the distribution of other Candida species reflecting the trends and antifungal susceptibilities. Previous studies have demonstrated that C. glabrata more frequently presents fluconazole-resistant strains. Extracts from Mexican plants have been reported with activity against pulmonary mycosis, among them Colubrina greggii . In the present study, extracts from the aerial parts (leaves, flowers, and fruits) of this plant were evaluated against clinical isolates of several species of Candida ( C. albicans , C. glabrata , C. parapsilosis , C. krusei , and C. tropicalis ) by the broth microdilution assay. Through bioassay-guided fractionation, three antifungal glycosylated flavonoids were isolated and characterized. The isolated compounds showed antifungal activity only against C. glabrata resistant to fluconazole, and were non-toxic toward brine shrimp lethality bioassay and in vitro Vero cell line assay. The ethyl acetate and butanol extracts, as well as the fractions containing the mixture of flavonoids, were more active against Candida spp.

Candida albicans is a prevalent opportunistic human fungal pathogen for which treatment is limited to only four main classes of antifungal drugs, with the azole and echinocandin classes being used most frequently. Drug tolerance, the ability of some cells to grow slowly in supra-MIC drug concentrations, decreases the number of available treatment options. Here, we investigated factors affecting tolerance and resistance to ketoconazole in C. albicans. We found both temperature and the composition of growth medium significantly affected tolerance with little effect on resistance. In deletion analysis of known efflux pump genes, CDR1 was partially required for azole tolerance, while CDR2 and MDR1 were dispensable. Tolerance also required Hsp90 and calcineurin components; CRZ1 , which encodes a transcription factor downstream of calcineurin, was required only partially. Deletion of VMA11 , which encodes a vacuolar ATPase subunit, and concanamycin A, a V-ATPase inhibitor, abolished tolerance, indicating the importance of vacuolar energy transactions in tolerance. Thus, tolerance to ketoconazole is regulated by multiple factors, including physiological and genetic mechanisms. IMPORTANCE Due to the ever-expanding range of invasive medical procedures and treatments, invasive fungal infections now pose a serious global threat to many people living in an immunocompromised status. Like humans, fungi are eukaryotic, which significantly limits the number of unique antifungal targets; the current arsenal of antifungal agents is limited to just three frontline drug classes. Additional treatment complexities result from the development of drug tolerance and resistance, which further narrows therapeutic options; however, the difference between tolerance and resistance remains largely unknown. This study demonstrates that tolerance and resistance are regulated by multiple genetic and physiological factors. It is prudent to note that some factors affect tolerance only, while other factors affect both tolerance and resistance. The complex underlying mechanisms of these drug responses are highlighted by the fact that there are both shared and distinct mechanisms that regulate tolerance and resistance.

A newly synthetized series of N -phenacyl derivatives of 2-mercaptobenzoxazole, including analogues of 5-bromo- and 5,7-dibromobenzoxazole, were screened against Candida strains and the action mechanism was evaluated. 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanone ( 5d ), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichloro-phenyl)ethanone ( 5i ), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,4,6-trichlorophenyl)ethanone ( 5k ) and 2-[(5-bromo-1,3-benzoxazol-2-yl)sulfanyl]-1-phenylethanone ( 6a ) showed anti- C. albicans SC5314 activity, where 5d displayed MIC T = 16 µg/mL (%R = 100) and a weak anti-proliferative activity against the clinical strains: C. albicans resistant to azoles (Itr and Flu) and C. glabrata . Derivatives 5k and 6a displayed MIC P = 16 µg/mL and %R = 64.2 ± 10.6, %R = 88.0 ± 9.7, respectively, against the C. albicans isolate. Derivative 5i was the most active against C. glabrata (%R = 53.0 ± 3.5 at 16 µg/mL). Benzoxazoles displayed no MIC against C. glabrata . Benzoxazoles showed a pleiotropic action mode: ( 1 ) the total sterols content was perturbed; ( 2 ) 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(3,4-dichlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichlorophenyl)ethanol ( 8h - i ) at the lowest fungistatic conc. inhibited the efflux of the Rho123 tracker during the membrane transport process; ( 3 ) mitochondrial respiration was affected/inhibited by the benzoxazoles: 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-chlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanol 8c - d and 8i . Benzoxazoles showed comparable activity to commercially available azoles due to ( 1 ) the interaction with exogenous ergosterol, ( 2 ) endogenous ergosterol synthesis blocking as well as ( 3 ) membrane permeabilizing properties typical of AmB. Benzoxazoles display a broad spectrum of anti- Candida activity and action mode towards the membrane without cross-resistance with AmB; furthermore, they are safe to mammals.

The epidemiology of yeast infections and resistance to available antifungal drugs are rapidly increasing, and non- albicans Candida species and rare yeast species are increasingly emerging as major opportunistic pathogens. In order to identify new strategies to counter the threat of antimicrobial resistant microorganisms, essential oils (EOs) have become an important potential in the treatment of fungal infections. EOs and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities. We investigated the in vitro antifungal activity of nine commercial EOs such as Thymus vulgaris (thyme red), Origanum vulgare (oregano), Lavandula vera (lavender), Pinus sylvestris (pine), Foeniculum vulgare (fennel), Melissa officinalis (lemon balm), Salvia officinalis (sage), Eugenia caryophyllata (clove) and Pelargonium asperum (geranium), and some of their main components (α-pinene, carvacrol, citronellal, eugenol, γ-terpinene, linalool, linalylacetate, terpinen-4-ol, thymol) against non- albicans Candida strains and uncommon yeasts. The EOs were analyzed by GC-MS, and their antifungal properties were evaluated by minimum inhibitory concentration and minimum fungicidal concentration parameters, in accordance with CLSI guidelines, with some modifications for EOs. Pine exhibited strong antifungal activity against the selected non- albicans Candida isolates and uncommon yeasts. In addition, lemon balm EOs and α-pinene exhibited strong antifungal activity against the selected non- albicans Candida yeasts. Thymol inhibited the growth of all uncommon yeasts. These data showed a promising potential application of EOs as natural adjuvant for management of infections by emerging non- albicans Candida species and uncommon pathogenic yeasts.

Aims: The present study aimed to determine the microsatellite length polymorphism (MLP) genotypic patterns and antifungal susceptibility profiles of Candida albicans isolated from patients with candiduria., Methods and Results: DNA of 50 C. albicans isolates was used for molecular identification based on the ITS 1 -5.8s-ITS 2 region. MLP assays were performed to amplify three loci (EF3, CDC3 and HIS3), and PCR products were used for fragment analysis. Antifungal susceptibility tests were performed according to CLSI M27 4th ed guidelines. In all, 38 different genotypes were detected with the three polymorphic loci among C. albicans isolates, and only one genotype was homozygous. In comparison to other countries, our genotypes were divided into three clusters, two of which were linked to France and a third of which was linked to Austria. The genetic structures of the 50 C. albicans isolates revealed varied heterozygosity and significant Hardy-Weinberg equilibrium at the EF3 locus. Only one (2%) and four (8%) of isolates showed resistance to fluconazole and itraconazole, respectively. In C. albicans genotype G25, one (2%) of the isolates showed cross-resistance and non-wild-type resistance to posaconazole, itraconazole and fluconazole., Conclusion: MLP typing is a useful tool to analyse the genetic structure of C. albicans isolates. High genetic diversity (38 genotypes) was detected in the isolates tested here. Compared to isolates in other countries, the ones from our patients had a clear relationship with French and Austrian isolates., Significance and Impact of the Study: Iranian isolates of C. albicans have a distinct genotype and show similarities only with French and Austrian isolates., (© 2021 The Society for Applied Microbiology.)

Synthetic fungicides have been widely used to protect crops from fungal diseases. However, excessive use of synthetic fungicides leads to the generation of fungicide resistance in fungal pathogens. Recently, smart cargo delivery systems have been introduced for the construction of a pesticide delivery nanoplatform, benefiting from their controlled release performance. Herein, a fungal pathogen microenvironment-responsive supramolecular fungicide nanoplatform has been designed and constructed, using quaternary ammonium salt (Q)-modified mesoporous silica nanoparticles (MSN-Q NPs) as nanocarriers loaded with berberine hydrochloride (BH) and carboxylatopillar[5]arene (CP[5]A) as nanogates to form BH-loaded CP[5]A@MSN-Q NPs for effective inhibition of Botrytis cinerea . CP[5]A as nanogates can endow the fungicide nanoplatform with pH stimuli-responsive release features for the control of fungicide release. The loaded BH, as a natural plant fungicide, provides an ecofriendly alternative to synthetic fungicides for controlling B. cinerea . Interestingly, we use oxalic acid (OA) secreted by B. cinerea as a trigger so that BH can be released from the fungicide nanoplatform on demand under pathogen microenvironments for controlling B. cinerea . The experimental results indicate that the fabricated fungicide nanoplatform could effectively inhibit the mycelial growth and spore germination, providing a new way for the management of B. cinerea in actual application.

There is a worldwide concern regarding the antimicrobial resistance and the inappropriate use of antifungal agents, which had led to an ever-increasing antifungal resistance. This study aimed to identify the antifungal susceptibility of colonized Candida species isolated from pediatric patients with cancer and evaluate the clinical impact of antifungal stewardship (AFS) interventions on the antifungal susceptibility of colonized Candida species. Candida species colonization was evaluated among hospitalized children with cancer in a tertiary teaching hospital, Shiraz 2017-2018. Samples were collected from the mouth, nose, urine, and stool of the patients admitted to our center and cultured on sabouraud dextrose agar. The isolated yeasts identified by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). DNA Extracted and PCR amplification was performed using the ITS1 and ITS4 primer pairs and Msp I enzyme. The broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) for amphotericin B, caspofungin, and azoles. The prevalence of Candida albicans in the present study was significantly higher than other Candida species. Candida albicans species were completely susceptible to the azoles. The susceptibility rate of C. albicans to amphotericin B and caspofungin was 93.1% and 97.1%, respectively. The fluconazole MIC values of Candida albicans decreased significantly during the post-AFS period (P < 0.001; mean difference: 72.3; 95% CI of the difference: 47.36-98.62). We found that ‏52.5% (53/117) of the isolated C. albicans were azole-resistant before AFS implementation, while only 1.5% (2/102) of the isolates were resistant after implementation of the AFS program (P < 0.001). C. albicans fluconazole and caspofungin resistant rate also decreased significantly (P < 0.001) after implementation of the AFS program [26 (32.9%) versus 0 (0.0%) and 11 (10.9%) versus 1 (0.9%), respectively]. Besides, fluconazole use (p < 0.05) and fluconazole expenditure reduced significantly (about one thousand US$ per year) after the AFS program. Our results confirm the positive effect of optimized antifungal usage and bedside intervention on the susceptibility of Candida species after the implementation of the AFS program. C. albicans and C. glabrata exhibited a significant increase in susceptibility after the execution of the AFS program.

Candida albicans is the most common human fungal pathogen, causing infections that range from mucous membranes to systemic infections. The present article provides an overview of C. albicans, with the production of biofilms produced by this fungus, as well as reporting the classes of antifungals used to fight such infections, together with the resistance mechanisms to these drugs. Candida albicans is highly adaptable, enabling the transition from commensal to pathogen due to a repertoire of virulence factors. Specifically, the ability to change morphology and form biofilms is central to the pathogenesis of C. albicans. Indeed, most infections by this pathogen are associated with the formation of biofilms on surfaces of hosts or medical devices, causing high morbidity and mortality. Significantly, biofilms formed by C. albicans are inherently tolerant to antimicrobial therapy, so the susceptibility of C. albicans biofilms to current therapeutic agents remains low. Therefore, it is difficult to predict which molecules will emerge as new clinical antifungals. The biofilm formation of C. albicans has been causing impacts on susceptibility to antifungals, leading to resistance, which demonstrates the importance of research aimed at the prevention and control of these clinical microbial communities., (© 2020 The Society for Applied Microbiology.)